Wulingsan Alleviates MAFLD by Activating Autophagy via Regulating the AMPK/mTOR/ULK1 Signaling Pathway

Here, we presented the study of the molecular mechanisms underlying the action of Wulingsan (WLS) in rats with metabolic-associated fatty liver disease (MAFLD) induced by a high-fat diet (HFD). High-performance liquid chromatography was employed to identify the chemical components of WLS. After 2 weeks of HFD induction, MAFLD rats were treated with WLS in three different doses for 6 weeks, a positive control treatment or with a vehicle. Lipid metabolism, liver function, oxidative stress, and inflammatory factors as well as pathomorphological changes in liver parenchyma were assessed in all groups. Finally, the expressions of autophagy-related markers, adenosine monophosphate-activated protein kinase (AMPK)/mechanistic target of rapamycin (mTOR)/unc-51-like kinase-1 (ULK1) signaling pathway-related genes, and proteins in liver were detected. The results revealed that WLS significantly ameliorated liver injury, the dysfunction of the lipid metabolism, the oxidative stress, and overall inflammatory status. Furthermore, WLS increased the expressions of LC3B-II, Beclin1, p-AMPK, and ULK1, along with decreased p62, p-mTOR, and sterol regulatory element-binding protein-1c levels. In conclusion, we showed that WLS is capable of alleviating HFD-induced MAFLD by improving lipid accumulation, suppressing oxidative stress and inflammation, and promoting autophagy.


Introduction
Nonalcoholic fatty liver disease (NAFLD) has become the leading cause of chronic liver disease, and it was recently defned as metabolic-associated fatty liver disease (MAFLD) to emphasize the role of metabolic dysfunction in this condition [1].MAFLD manifests as hepatic steatosis in combination with overweight/obesity, type 2 diabetes, or metabolic dysregulation.It afects approximately 25.24% adults worldwide, with the highest prevalence rates in the Middle East and South America [2,3].Moreover, the prevalence of MAFLD also increases among children and young adolescents [4].MAFLD can progress to nonalcoholic steatohepatitis (NASH), leading to hepatic fbrosis and hepatocellular carcinoma [5].Furthermore, MAFLD is associated with an increased risk of type 2 diabetes, cardiovascular disorders, and certain cancers, further exacerbating the health and economic burden on society [6,7].However, the available pharmacological therapies for this disease remain limited.
Although the development of MAFLD is a complex and multifactorial process, the classical "two-hit" theory suggested that oxidative stress and infammation play crucial roles in its pathogenesis.Te "frst hit" is characterized by hepatic lipid accumulation and insulin resistance.Subsequently, the "second hit" emerges, involving infammatory factors, mitochondrial dysfunction, and oxidative stress, which further exacerbate MAFLD and lead to NASH.Recent studies have increasingly suggested the relevance of autophagy in delaying the progression of MAFLD [8].Autophagy, a lysosomal degradation pathway, reduces hepatic lipid deposition, improves insulin resistance, and inhibits oxidative injury and infammation [9].Lipid metabolism disorder is a key link in the pathogenesis of MAFLD, and autophagy plays a crucial role in regulating hepatic lipid metabolism [10,11].Lipophagy, a selective autophagic process for the catabolism of lipid droplets in the lysosomes, supplies free fatty acid (FFA) to produce energy and ketone bodies in hepatocytes through β-oxidation [12].Research has shown that activation of autophagy could signifcantly inhibit hepatic steatosis, while defective autophagy may lead to hepatic steatosis and further accelerate the development and progression of MAFLD [13].Adenosine monophosphate-activated protein kinase (AMPK), an essential energy regulator, promotes autophagy by inhibiting the mechanistic target of rapamycin (mTOR) and activating unc-51-like kinase-1 (ULK1) [14].Additionally, phosphorylated AMPK decreases the expression of sterol regulatory element-binding protein-1c (SREBP-1c), thereby reducing adipogenesis [15].Activating AMPK/ULK1 signaling and inhibiting mTOR phosphorylation could reduce lipid accumulation in high-fat diet (HFD)-induced obese mouse [16].However, FFA-induced HepG2 cells were treated with AMPK inhibitor Compound C (CC), or silence of ULK1 by transfection with siRNA could inhibit hepatic autophagy and promote lipid accumulation [17].Tese fndings demonstrated that targeting the AMPK/mTOR/ULK1 signaling pathway and promoting autophagy may ofer new therapeutic strategies for MAFLD.
In China, traditional Chinese medicine (TCM) has become widespread for over 2000 years.TCM follows the principle of "multiple ingredients-multiple targetsmultiple pathways" and holds promise as a novel approach for treating MAFLD.One TCM prescription, Wulingsan (WLS), has traditionally been used to regulate body fuid homeostasis.Its frst mention can be traced back to the third century in the Shanghan Lun, written by Zhang ZhongJing.WLS is composed of zhuling (Polyporus), fuling (Poria cocos), zexie (Alismatis rhizoma), baizhu (Atractylodis macrocephalae rhizoma), and guizhi (Cinnamomi ramulus), and it is known for its efcacy in warming the yang, strengthening the spleen, and eliminating dampness to address phlegm-related issues.In TCM, MAFLD is often attributed to spleen dysfunction and the accumulation of "phlegm-dampness."Although "phlegm-dampness" is an abstract concept in the TCM system, we understand its close association with body fuid homeostasis.Modern research has demonstrated that WLS could regulate lipid metabolism in obese rats [18].Various extracts and bioactive monomers of WLS have been found to ameliorate infammation and oxidative stress.Polyporus polysaccharide induced autophagy to inhibit the proliferation of cancer cell [19], and reduced infammation in lung and kidney fbrosis mouse [20,21].Poria cocos polysaccharide protected liver injury by suppressing infammation and oxidative stress [22].In addition, Poria cocos extract also alleviated HFD-induced MAFLD by regulating lipid homeostasis and bile acid metabolism [23].Alisol A 24-acetate, a triterpene from Alismatis rhizoma, could ameliorate hepatic steatosis and inhibit infammation [24].Atractylodis macrocephalae rhizoma could promote energy metabolism to prevent obesity [25].Furthermore, essential oil from Atractylodis macrocephalae rhizoma has antioxidant and antiinfammatory activities [26].Cinnamomi ramulus essential oil showed anti-infammatory efects in Nacylethanolamine acid amidase-overexpressing HEK293 cells [27].However, it is unclear whether WLS could activate autophagy to suppress oxidative stress and infammation and thus ameliorate MAFLD.Terefore, this study aimed to explore the protective efects of WLS on MAFLD rats induced by HFD and elucidate the role of WLS in regulating the AMPK/mTOR/ULK1 signaling pathway.

High-Performance Liquid Chromatography (HPLC)
Analysis.Te main chemical components of WLS were analyzed using HPLC (Essentia LC-16 system, SHI-MADZU, Tokyo, Japan), and chromatographic separation was conducted on a Shim-pack GIST C18 column After 1 week of acclimatization, the rats were randomly divided into two groups: the control group (n � 10) and the HFD group (n � 50).Te control group received drinking water and standard chow, while the HFD group was given a HFD (78.8% basic feed, 15% lard, 5% sucrose, 1% cholesterol, and 0.2% sodium cholate) for 8 weeks.Following 2 weeks of HFD feeding, the rats in the HFD group were further divided into fve subgroups (n � 10): the model group received normal saline via gavage (10 mL/kg), the PPC group received PPC (0.144 g/kg) as a positive control, and the WLS groups were administered diferent doses of WLS (0.777, 1.554, and 3.108 g/kg, equivalent to 1, 2, and 4 times the clinically equivalent dose, referred to as L-WLS, M-WLS, and H-WLS, respectively).Each group of rats were treated by gavage at 8:30 a.m.daily.Te dosage of drugs was calculated according to "Methodology on Chinese medicinal pharmacology" [28].
After 6 weeks of treating, rats were anesthetized with an intraperitoneal injection of 3% sodium pentobarbital at a dose of 1 mL/kg.Ten, blood and liver were rapidly collected.

Biochemical Analysis. Serum lipids (TC, TG, HDL-C, and LDL-C), liver function indicators (ALT and AST
), and liver lipids (TC, TG, and FFA) were determined using a biochemical analysis (Humalyzer 2000, Germany).Enzyme-linked immunosorbent assay (ELISA) was employed to assess the serum levels of IL-1β and IL-6.Te activities or levels of SOD, CAT, GSH, and MDA in serum were measured using respective kits according to the manufacturer's instructions.

Liver Histopathological Evaluations.
Liver tissues were fxed in 4% paraformaldehyde, embedded in parafn, dehydrated, and sliced for hematoxylin and eosin (H&E) staining.At the same time, NAFLD activity score (NAS) was used to score the liver histomorphology [29], and the specifc scoring criteria are shown in Table 1.
Additionally, 5-μm frozen liver sections were stained with oil red O staining.Te stained sections were observed under a microscope (Nikon Eclipse E100, Tokyo, Japan).Te staining area (Area) and integral absorbance (IA) of oil red O were determined using Image Pro Plus, and IOD (sum)/Area (sum) was used as the fnal result.

Quantitative Real-Time PCR (qRT-PCR) Analysis.
Total RNA was extracted from liver tissues using Trizol reagent and reverse-transcribed into cDNA using a Tian-Script RT reagent kit.Te primer sequences are provided in Table 2. Amplifcation was performed under the following conditions: 95 °C for 15 min, 40 cycles of 95 °C for 10 s, and 58 °C for 60 s.Te expressions of genes were analyzed using the 2 −ΔΔCt method.

Immunohistochemistry Staining (IHC).
Te expressions of p62, ULK1, and SREBP-1c in liver were assessed using IHC.Liver tissues fxed in 4% paraformaldehyde were embedded in parafn, dehydrated, subjected to antigen retrieval, and treated with 3% H 2 O 2 to block endogenous peroxidase activity.Te tissues were then blocked with 3% BSA and incubated overnight at 4 °C with the primary antibody (1 : 500), followed by incubation with the secondary antibody (1 : 500).Subsequently, the slides were stained using a DAB color developing solution and counterstained with hematoxylin.Te stained slides were observed under a microscope, and the presence of brown-yellow staining was considered as positive expression.Image Pro Plus 6.0 software was used to measure the integrated optical density value (IOD) and pixel area (Area) of each image, and the ratio of IOD (sum)/Area (sum) was calculated.
2.9.Western Blot Analysis.Te liver tissues were homogenized in 1 mL RIPA lysis bufer and 10 μL PMSF for extracting protein.Te extracted proteins were then separated by 10% SDS-PAGE and transferred onto a PVDF membrane.Te membrane was blocked with a rapid closure solution for 5 min and incubated overnight at 4 °C with specifc primary antibodies (1 : 1000) against AMPK, p-AMPK, mTOR, p-mTOR, LC3B, and Beclin1.Afterward, the membrane was incubated with the secondary antibody (1 : 5000) for 30 min.Protein bands on the membrane were visualized using an enhanced chemiluminescence (ECL) reaction and detected using a multifunctional imaging system (Fusion FX5 Spectra, Vilbe, France).Te expressions of proteins were analyzed using Image J.

Statistical Analysis.
Te data were presented as mean ± standard deviation (SD) and analyzed using SPSS 19.0 (IBM, Armonk, NY, USA).One-way analysis of variance (ANOVA) was performed to analyze the data, followed by a Tukey's test or Dunnett'T3 test for post hoc comparisons.P < 0.05 was considered statistically signifcant.

Identifcation of Chemical Components in WLS by HPLC.
As shown in Figure 1, a total of 7 chemical components were determined in WLS, named ferulic acid, cinnamic acid, cinnamaldehyde, alisol C-23-acetate, atractylenolide I, atractylenolide III, and atractylenolide II.Te contents of these components are presented in Table 3.To perform linear regression analysis, standard curves of peak area (Y) and concentration (X) of the standards were plotted.As indicated in Table 3, 7 components exhibited a strong linear relationship under the given chromatographic conditions.

WLS Attenuated Liver Injury in MAFLD
Rats.In the model group, a long-term HFD resulted in severe liver damage, leading to a signifcant increase in serum ALT and AST activities.Following the treatment of MAFLD rats with various doses of WLS, a noticeable reduction in serum ALT and AST activities was observed.As shown in Figures 2(c) and 2(d), more lipid droplet vacuoles and ballooning were observed, and NAS was notably increased in the model group.Te hepatic pathological damage and NAS were obviously improved after the treatment of WLS and PPC.

WLS Regulated Lipid Metabolism in MAFLD Rats.
Next, we employed oil red O staining to examine hepatic lipid deposition.As illustrated in Figures 3(a) and 3(b), the model group exhibited a signifcant increase in red lipid droplets compared to the control group.However, the WLS groups demonstrated a marked reduction in hepatic lipid accumulation.
Te lipid levels in both serum and liver are presented in Figures 3(c)-3(i).In the model group, there was an elevation in serum lipids (TC, TG, and LDL-C) and hepatic lipids (TC, TG, and FFA).Conversely, the serum HDL-C level was decreased.However, treatment with WLS efectively reduced the levels of serum lipids (TC, TG, and LDL-C) and hepatic lipids (TC, TG, and FFA) while increasing serum HDL-C levels.

WLS Suppressed Oxidative Injury and Infammation in MAFLD
Rats.In the model group, there was the notable decrease in the activities or contents of SOD, CAT, and GSH in serum.Additionally, the levels of MDA, IL-1β, and IL-6 in serum were signifcantly increased.However, following 6 weeks of WLS treatment, there was a marked increase in the activities or contents of SOD, CAT, and GSH in serum, accompanied by a signifcant decrease in MDA, IL-1β, and IL-6 levels in serum (Figure 4).

WLS Ameliorated Lipid Deposition, Induced Autophagy,
and the Activated AMPK/mTOR/ULK1 Signaling Pathway in MAFLD Rats.Subsequently, we assessed the levels of autophagy markers (Beclin1, LC3B, and p62) through western blot, qRT-PCR, and IHC to investigate the impact of WLS on autophagy in MAFLD rats (Figure 5).In the model group, the expressions of Beclin1 and LC3B-II were markedly downregulated, while p62 expression was significantly upregulated.However, following WLS treatment, there was a notable increase in the levels of Beclin1 and LC3B-II proteins, accompanied by a decrease in p62 expression.Canadian Journal of Gastroenterology and Hepatology To ascertain whether WLS regulated the AMPK/mTOR/ ULK1 signaling pathway to mediate autophagy, we evaluated the protein levels of AMPK, p-AMPK, mTOR, p-mTOR, and ULK1 in the liver.As depicted in Figure 6, the results demonstrated that the p-AMPK/AMPK and ULK1 expressions were decreased, whereas the p-mTOR/mTOR was elevated in the model group.However, WLS not only upregulated the p-AMPK/AMPK and ULK1 expressions but also downregulated the p-mTOR/mTOR.Additionally, qRT-PCR results indicated that WLS enhanced the expression of ULK1.Canadian Journal of Gastroenterology and Hepatology Next, we examined the expression of SREBP-1c, a downstream factor of AMPK known to regulate lipid synthesis.Notably, WLS treatment led to a signifcant decrease in both the gene and protein expressions of SREBP-1c (Figure 7).Collectively, these fndings indicate that WLS reduced lipid deposition and activated autophagy by modulating the AMPK/mTOR/ULK1 signaling pathway.

Discussion
In TCM, the spleen is responsible for transporting and transforming water and dampness.A sedentary lifestyle, irregular diet, or emotional disorders can lead to spleen dysfunction in transportation and the accumulation of dampness and phlegm, resulting in the occurrence of MAFLD.Te formulation characteristics of WLS are consistent with the pathogenesis of spleen defciency and phlegm-dampness in MAFLD.Zexie and zhuling could promote urination and drainage, fuling and baizhu have the efect of strengthening the spleen and removing dampness, and guizhi could warm the spleen yang and strengthen the spleen qi.Terefore, WLS may efectively strengthen the spleen, drain dampness, and eliminate phlegm.WLS has been used for thousands of years to treat renal disease [30,31].Clinically, WLS has been reported to signifcantly decrease lipid content [18].Lipid metabolism disorder is the central link of the occurrence and progression of MAFLD.Modern medicine has found that dyslipidemia can be used as the microscopic manifestation of phlegm-dampness syndrome [32].Te serum levels of TC and TG in patients with phlegmdampness syndrome were signifcantly higher than those in patients without phlegm-dampness syndrome, and the degree of hepatic lipid accumulation was directly proportional to the degree of phlegm dampness [33,34].Longterm HFD can cause severe liver injury, and the activities of ALT and AST in serum are the most sensitive indicators to evaluate the degree of liver injury.In our study, HFD increased the levels of serum lipids (TC, TG, and LDL-C) contents, hepatic lipids (TC, TC, FFA) contents, as well as Autophagy is a cellular self-protection program activated during stress responses involving the recycling of harmful proteins and organelles through the lysosomal degradation pathway [35,36].Emerging evidence suggested that autophagy plays a role in regulating lipid metabolism through lysosome degradation [9].Previous studies have demonstrated that inhibiting the expression of autophagyassociated proteins impairs the autophagy process, leading to the accumulation of lipid droplets and TG in liver, ultimately accelerating liver injury [37,38].Conversely, enhancing autophagy could improve mitochondrial function and mitigate the lipotoxicity caused by palmitic acid in mouse hepatocytes [39].Terefore, promoting the activation of autophagy may serve as a potential strategy to regulate lipid metabolism in liver.
Autophagy is a continuous process, and Beclin1, p62, and LC3B are commonly used markers to assess autophagy dynamics [40].Beclin1 is a key protein involved in regulating the initiation and nucleation of autophagosome formation, and its expression is positively correlated with autophagy [41].Studies have shown that decreased Beclin1 expression disrupts autophagosome degradation in cultured cells [42].LC3 consists of three isoforms: LC3A, LC3B, and LC3C, with LC3B being a highly conserved gene in mammals.During autophagosome formation, the plasma membrane-associated LC3B-I is conjugated with phosphatidylethanolamine at its C-terminus, forming LC3B-II [43].Terefore, an increase in LC3B-II indicates autophagy activation and an increase in the number of autophagosomes.During autophagy, p62 binds to ubiquitinated proteins in the cytoplasm and subsequently interacts with LC3B-II on the autophagosome membrane.Te complex is then degraded in autophagolysosomes [41].Accumulation of p62 is a well-known characteristic of autophagy defciency [44].In our studies, we observed a decrease in LC3B-I to LC3B-II conversion and Beclin1 expression, as well as an upregulation of p62 in MAFLD rats.Conversely, treatment with WLS increased LC3B-II and Beclin1 expressions while decreasing p62 levels.Tese results indicated that WLS promotes autophagy in HFDinduced MAFLD rats.
AMPK/mTOR/ULK1-mediated autophagy activation has a positive efect on treating MAFLD.Autophagy is amplifed by AMPK, a pivotal factor in maintaining energy homeostasis [45].AMPK is a heterotrimeric protein consisting of α, β, and c subunits.Te regulatory subunits include β and c subunits, while the catalytic subunit is the α subunit.Te phosphorylation of threonine 172 residue on the α subunit is essential for AMPK activation [46].mTOR  10 Canadian Journal of Gastroenterology and Hepatology can form TOR complex 1 (TORC1) and TOR complex 2 (TORC2) by interacting with various binding partners.Te activation of mTORC1 suppresses autophagy [47,48] Additionally, ULK1, an autophagy-initiating kinase, plays a pivotal role in autophagy initiation [49].It is well established that AMPK and mTOR are essential for activating ULK1 to initiate autophagy [50].On the one hand, mTORC1 phosphorylates Ser757 of ULK1, leading to the inhibition of its activity [17].On the other hand, phosphorylated AMPK inactivates mTORC1 by phosphorylating tuberous sclerosis 2 and Raptor protein within mTORC1.Importantly, AMPK can directly activate ULK1 by phosphorylating Ser555, Ser317, and Ser777 of ULK1 [51].SREBP-1c is mainly expressed in hepatocytes and adipocytes, and is a vital transcription factor that regulates lipogenesis by activating genes involved in fatty acid and triglyceride synthesis.Phosphorylated AMPK can phosphorylate the Ser372 residue of SREBP-1c to inhibit the expression of SREBP-1c, reducing fatty acid synthesis and lipid accumulation, and improving hepatic steatosis [52].Our data demonstrated that WLS signifcantly phosphorylated AMPK and activated ULK1.Simultaneously, WLS treatment suppressed the activation of mTOR and SREBP-1c.Tese results collectively indicated that WLS alleviates HFD-induced MAFLD by regulating the AMPK/mTOR/ULK1 signaling pathway to promote autophagy and reduce lipid accumulation.Meanwhile, the activation of autophagy also helps reduce oxidative stress and infammatory responses.Autophagy plays a role in regulating the secretion of infammatory cytokines.Studies have shown that the induction of autophagy pathways dependent on LC3B and p62 inhibits IL-1β and IL-6 production [53,54].IL-1β is mainly produced by hepatic macrophages and plays a major role in promoting insulin resistance, hepatic lipid accumulation, and inducing hepatic fbrosis [55].IL-6 has chemotactic activities, which can promote local migration and infltration of infammatory cells into the fatty liver, thereby facilitating the cascade amplifcation of the infammatory response in liver [56].Further, autophagy mitigates oxidative stress by eliminating peroxides from damaged mitochondria and repairing damaged DNA [57,58].SOD, a metalloproteinase, can convert oxygen free radicals into hydrogen peroxide and molecular oxygen [59].Hydrogen peroxide is harmful for body and could be immediately catalysed by CAT into completely harmless water [60].GSH is a free radical scavenger, its content will lead to the accumulation of oxygen free radicals, increase the oxidative stress.Tus, the SOD, CAT and GSH activities indicates the capacity of the body's antioxidant.MDA as the production of lipid peroxidation, is regarded as oxidative stress of toxic molecules and biological markers.In our research, the administration of WLS signifcantly decreased the contents of IL-1β, IL-6, and MDA while increased SOD, GSH, and CAT activities and levels.Tese results demonstrated the antioxidant and antiinfammatory efects of WLS.However, the mechanism of WLS activates autophagy to inhibit oxidative stress and infammation reaction has not been clarifed, which needs to be studied further.

Conclusion
In conclusion, our fndings provided evidence that WLS can alleviate HFD-induced MAFLD by reducing lipid deposition and suppressing oxidative stress and infammation through activating autophagy via regulating AMPK/mTOR/ULK1 signaling pathway.Tese results ofered novel experimental evidence supporting the potential of targeting AMPK/ mTOR/ULK1 signaling pathways as a therapeutic approach for MAFLD.

Figure 3 :Figure 4 :
Figure 3: Efect of WLS on lipid metabolism in MAFLD rats.(a) Oil red O staining of liver tissue (×200).(b) Quantifcation of oil red O positive area.(c-f ) Te levels of TC, TG, LDL-C, and HDL-C in serum.(g-i) Te levels of TC, TG, and FFA in the liver.Results were expressed as the mean ± SD, n � 3 in a-b, n � 10 in (c-i).* P < 0.05, * * P < 0.01, compared with the model group.

Figure 6 :
Figure 6: Efect of WLS on regulating the AMPK/mTOR/ULK1 signaling pathway in MAFLD rats.(a-c) Hepatic p-AMPK, p-mTOR proteins expressions.(d-f) Hepatic ULK1 protein and gene expression.Results were expressed as the mean ± SD, n � 3 in (a-e) and n � 6 in (f ) * P < 0.05 and * * P < 0.01, compared with the model group.

Figure 7 :
Figure 7: Efect of WLS on the expressions of SREBP-1c in MAFLD rats.(a-c) Expression of protein and gene of SREBP-1c in the liver.Results were expressed as the mean ± SD, n � 3 in (a, b) and n � 6 in (c).* P < 0.05 and * * P < 0.01, compared with the model group.

Table 3 :
Detection wavelength, linear regression data, regression data, and content of 7 constituents in WLS.
Canadian Journal of Gastroenterology and Hepatology